In vivo monitoring method of transgenic plants and system using the same

ABSTRACT

The present invention relates to a method for visualizing GFP expression in callus, various tissue and organ of the transgenic plants as image and system using the same. The said method needs no other additional genetic product, substrate or cofactor and can detect very simply and quickly GFP expression by using the said system of the present invention consisting of a CCD camera, a light source, band-pass filter and data processing computer, so it provides many advantages for selection of transgenic seeds, for studying of gene expression in the tissue or organ of plants, or for studying of specificity of each development step.

TECHNICAL FIELD

[0001] The present invention relates to a method and system formonitoring the transformation of plants. More particularly, the presentinvention relates to a fluorometry method and system for thevisualization of the in vivo expression of a green fluoroscent proteingene tagged to an exogenous gene of interest, thereby enabling theplants to be determined as to whether they are transformed with theexogenous gene or not.

BACKGROUND ART

[0002] Reporter proteins are usually used as markers for visualizing invivo gene expression and protein translocation in eucaryotic andprokaryotic organisms. The most widely used reporter proteins in plantsmay be exemplified by β-glucuronidase (GUS) and luciferase (LUC).Particularly, GUS is a prevalent visualization marker in the plant cellbiology (Jefferson R A, et al., (1987), EMBO J., 6:3901-3907). However,histochemical GUC analysis is not suitable for the direct visualselection of transgenic plants per se because it requires thedestruction of plant tissues. As for LUC, its in vivo synthesis can bedetected; however, an external substrate, that is luciferin, is requiredfor the detection of LUC. In addition, LUC has the disadvantage ofemitting a low intensity of light (Ow D W et al., Science 234:856-859).

[0003] Recently, it has been reported that the green fluorescent protein(GFP) of jellyfish Aquorea Victoria can be utilized as a sensitivereporter for in vivo gene expression (Chalfie M., et al., 1994, Science263:802-805). Even though requiring no external factors, the detectionof the fluorescence generated from an isolated GFP is possible only withnear infrared (386 nm) or blue light (475 nm). However, the fluorescenceof the GFP can be observed under visible light in room conditions(Chalfie M., et al., 1994, Science 263:802-805; Delagrave S., et al.,1995, Bio/Technology 13:151-154; Heim R, et al., 1994, Proc Nat'l, AcadSci. USA, 94:2122-2127). Further, GFP model is very advantageous in thatit retains fluorescence even when being expressed in heterologousbiosystems, such as E. coli, yeast, Drosophila, insects, mammals, etc.(Brand A 1995, TIG 11:324-325; Chalfie M., et al., 1994, Science263:802-805; Cubitt A B, et al., 1995, TIBS 20:448-455; Davis S J, etal., 1998, Plant Mol. Biol. 36:521-528; Delagrave S, et al., 1995,Bio/Technology 13:151-154; Haseloff J., et al., 1997, Proc. Nat'l Acad.Sci. USA, 94:2122-2127; Rosario R., et al., 1995, Curr. Biol. 5:635-642;Wang S X et al., 1994, Nature 369:400-403).

[0004] Bioassays for gene expression using fluorescence are very usefulfor monitoring the transformation and growth of plants. Because of planttissues' being composed of highly reflective cell walls and aqueouscytoplasm containing various autofluorescent and light-scatteringmaterials, it is difficult to directly observe the exogenousfluorescence of proteins introduced into plant tissues under afluorescent optical microscope (Haseloff J. et al., 1998, Greenfluorescent protein: Properties, applications, and protocols. ChalfieM., Kain S., Eds., Wiley-Liss, New York, pp 191-242). For this reason,the use of GFP as a marker for the selection of transgenic plants underdirect visual conditions has not yet been reported.

DISCLOSURE OF THE INVENTION

[0005] Leading to the present invention, the intensive and thoroughresearch on the identification of transgenic plants, conducted by thepresent inventors, resulted in the finding that, when a filtered lightbeam which can excite GFP is projected to a plant sample of interest,light is emitted from the plant sample and, if filtered through a greenbandpass filter, can be analyzed for the expression of GFP by use of aCCD (charge-coupled device) camera imaging system.

[0006] Therefore, it is an object of the present invention to provide amethod for monitoring the transformation of plants using GFP as areporter.

[0007] It is another object of the present invention to provide a CCDimaging system for visualizing the in vivo expression of GFP.

[0008] In an aspect of the present invention, there is provided afluorometry method for monitoring the transformation of plants based onthe in vivo expression of a heterologous green fluorescent protein,comprising the steps: projecting excitation light from a light sourcethrough a blue bandpass filter onto a plant sample at an angle of 45°,said blue bandpass filter passing light ranging, in wavelength, from 470to 490, said excitation light having a wavelength of around 488 nm witha peak at 480 nm; detecting light generated from the plant sample by useof a charge coupled device color video camera equipped with a zoom lens,which is positioned on the axis vertical to the plane of the sample,said light passing through a green bandpass filter which passes lightranging, in wavelength, from 500 to 550 nm before arriving at said zoomlens, so as to have a wavelength of around 509 nm, and photographing theimage of the plant sample on the basis of the light generated from theplant sample; and processing the image in a computer to determinewhether the plant sample is transgenic or not.

[0009] In another aspect of the present invention, there is provided asystem for monitoring the transformation of plants on the basis of thein vivo expression of a heterologous green fluorescent protein,utilizing the method.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

[0011]FIG. 1 is a schematic diagram showing the plasmid pSBG700.

[0012]FIG. 2 shows excitation (A) and emission (B) spectra of GFP inwild type and transgenic rice leaves.

[0013]FIG. 3 is an illustration of a CCD imaging system for GFP.

[0014]FIG. 4 shows profiles of light beams which pass through bandpassfilters established in front of a light source and a CCD lens,respectively.

[0015]Fig. 5 shows fluorographs visualizing the expression of GFP in awild type rice callus (A) and a transgenic rice callus (B).

[0016]FIG. 6 shows fluorographs visualizing the expression of GFP in awild-type, white rice sprout (A), a transgenic, white rice sprout (B), awild-type, green rice sprout (C), and a transgenic, green rice sprout(D).

[0017]FIG. 7 shows fluorographs visualizing the expression of GFP in awild-type, unshelled rice seed (A), a transgenic, unshelled rice seed(B), and a transgenic, shelled rice seed (C).

[0018]FIG. 8 is a schematic diagram illustrating the tagging of a gfpgene to a gene of interest and the crossing of a transformed line with awild type line.

[0019]FIG. 9 shows a seed sorting machine useful to carry out anembodiment of the present invention.

BEST MODES FOR CARRYING OUT THE INVENTION

[0020] The present invention pertains to the non-invasive visualizationof the fluorescence of the GFP expressed in transgenic plant tissues ororgans, including rice calluses grown in light-illuminated and shieldedconditions, with the aid of a fluorometry system comprising a CCDcamera, a light source, bandpass filters, and a computer for processingdata, in accordance with the present invention.

[0021] In the present invention, a bioassay using fluorescence of GFP isprovided for monitoring the transformation of plants. Fluorometry fordetecting in vivo fluorescence of the GFP expressed in transformedplants and a system therefor are described in connection with theaccompanying drawings. Before the present method and system formonitoring the transformation of plants is disclosed or described, it isto be understood that the terminology used therein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting.

[0022] The application of the preferred embodiments of the presentinvention is best understood with reference to the accompanyingdrawings.

[0023] Referring to FIG. 3, there is shown the application of a digitalvideo imaging system to a leaf to identify whether the plant istransgenic or not by detecting the fluorescence emitted therefrom. As alight source, there is used a 250W halogen lamp 1 which generatesexcitation light. In front of the halogen lamp 1 is positioned a bluebandpass filter 2 (Edmund, USA) through which only the light with awavelength in the range of 470 to 490 nm (max light wave 480 nm) canpass, because light with a wavelength of around 488 nm can excite GFP.Light beams are projected to a plant sample 3 at an angle of about 45°to the axis vertical to the plane of the plant sample. In one embodimentof the present invention, the illumination area was set to be 4 cm² at amaximum photon flux density (PFD) of 2 μmole m⁻²s⁻¹. When the filteredlight is irradiated to the sample, the GFP expressed in vivo is excitedand emits fluorescent light. Using this fluorescence generated from thesample, the image of the sample can be visualized in a photograph. Inthis regard, a high-resolution CCD color video camera 6 equipped with azoom lens 5 is positioned on the axis vertical to the plane of thesample to take a photograph of the sample while focusing on a certainarea of the sample with the zoom lens. Because the light emitted fromthe GFP has a wavelength of about 509 nm, a bandpass filter 4 forpassing the green light ranging in wavelength from 500 to 550 nm isestablished in front of the CCD camera to pass the green fluorescentlight only. Then, images photographed by the CCD are sent to a computerin which the image data are collected, stored and processed.

[0024] With reference to FIG. 4, there are shown transmittances of thelight having peaks at 480 nm and 510 nm, which are filtered through theblue bandpass filter 2 and the green bandpass filter 4, respectively.

[0025]FIG. 9 shows a sorting machine for separating seeds transformedwith a gfp gene from those that lack the exogenous gfp gene by takingadvantage of the fluorescence resulting from the in vivo expression ofGFP. Seeds having an exogenous gene tagged with a gfp gene can beobtained as illustrated in FIG. 8. A gene which provides acharacteristic of interest is combined with a gfp gene to give a hybridDNA, followed by the transduction of the hybrid DNA into a plant cellline A. Then, the plant seed harboring the gene of interest is crossedwith a cell line B which does not contain the exogenous gene to producea gfp-transformed progeny F1. Returning to FIG. 9, harvested seeds 4′are put in a seed collector 1′ and dropped onto a conveyor belt 3′through a seed path 2′ which restricts the size of the seeds 4′. Theseeds 4′ arrived on the conveyor belt 3′ are moved as the conveyor belt3′ runs. During the migration on the conveyor belt 3′, the seeds 4′ passby a fluorometry system and a seed sorter. At this time, light isirradiated from a light source 5′ to a seed 4′. In this connection, thelight generated from the light source 5′ is filtered through a bandpassfilter 6′ so that the wavelengths of the light illuminated on the seed4′ fall within a particular range. When being irradiated with a lightbeam within a particular wavelength range, the seed 4′ fluoresces if itis transformed with the gene of interest tagged with the gfp gene. Then,the fluorescence is detected by a CCD camera 9′ equipped with a zoomlens 8′, which is established to receive the fluorescent light in thedirection perpendicular to the migration direction of the conveyor belt3′. Before entering the zoom lens 8′, the fluorescence light generatedfrom the seed 4′ goes through a bandpass filter 7′ which passes thelight in the wavelength range of GFP fluorescence only. An image of theseed is visualized in the CCD camera 9′ from which the image informationis transmitted to the data processor 10′ in which the information isprocessed to determine whether the seed is transgenic or not. A signal11′ concerning the determination is generated from the processor 10′ andsent to a controller 12′. According to the signal, the controller 12′operates a seed sorter 13′ in ON/OFF states alternatively. For example,when the seed 4′ does not show fluorescence, the seed sorter 13′ is letto enter an ON state to select the seed 4′ into a non-transgenic seedcollector 14′. On the other hand, when the fluorescence is detected fromthe seed 4′, the operation of the seed sorter 13′ is ceased so that theseed 4′ is migrated, along the conveyor belt 3′, to a transgenic seedcollector 15′. Alternatively, the seed sorter 13′ may be operated insuch a way as to select transgenic seeds only. In this case,non-transgenic seeds 4′ are let to migrate to the end of the belt 3′.Therefore, the seed sorting machine of the present invention allows thepurity of the transgenic progeny F1 and the line B to be assayed, aswell as enabling the selection of the transgenic F1 progeny.

[0026] The method for detecting the fluorescence resulting from GFPexpression of the present invention enjoys the advantages of beingperformed quickly and with ease and requiring no additional factors,including gene products, substrates, subsidiary factors, etc. Forexample, it is possible to determine whether a protein of interest isexpressed in vivo with the aid of neither enzymes nor antibodies. Morerecently, there has been suggested the potential application of GFP as avisualization marker in tobacco plants (Molinier J. et al., 2000, PlantCell Reports 19:219-223). However, it was very difficult to take aphotograph of a green image from old leaves in which vacuoles are welldeveloped.

[0027] Based on the in vivo expression of GFP, the visualization methodof the present invention is anticipated to make great contribution tothe study of gene expression in various plant tissues and organs and ofdevelopmental traits, as well as the selection of transgenic seeds.

[0028] A better understanding of the present invention may be obtainedin light of the following examples which are set forth to illustrate,but are not to be construed to limit the present invention.

EXAMPLE 1 Construction of Vector

[0029] After being digested with BamHI and NcoI, a rice-derived Act1promoter (McElroy D. et al., 1991, Mol. Gen. Genet. 231:150-160 wasinserted into a BamHI/NcoI-linearized pBluescript plasmid containing ansgfp gene (Kohler R H. et al., 1997, Plant J. 11:613-621). Treatment ofthe resulting recombinant plasmid with BamHI and NotI extracted an Act1promoter-sgfp fragment. This DNA fragment was ligated to aBamHI/NotI-linearized pSB105 that contained potato proteinase inhibitorII terminator/35S promoter/bar/nopaline synthase terminator to constructa recombinant plasmid, named pSBG700, as illustrated in FIG. 1. Usingthe tripatental mating method disclosed in Komari T., et al., 1996,Plant J. 19:165-174), Agrobacterium tumefacience LBA4404 was transformedwith the plasmid pSBG700.

EXAMPLE 2 Transformation of Rice

[0030] A wild-type rice seed (Oryza sativa cv. Nakdong) and a rice seedtransformed with an agfp gene (Jang et al., 1999, Molecular breeding5:453-461) were treated with 70% ethanol for 1 hour and then with 10%sodium hypochlorite for 15 min. After being washed five times withsterile water, the seeds were sowed in test tubes containing MS salt(Murashige T., Skoog F., 1962, Physiol. Plant, 15:473-497), sucrose 30g/l, and bactoagar 8 g/l. Subsequently, the test tubes were incubated ina growth chamber which was adjusted to a temperature of 25° C. with aPFD maintained at 100 μmol m⁻²s⁻¹. Under light-illuminated and shieldedconditions, the seeds were let to grow for 16 hours and 8 hours,respectively. The seeds were germinated to white plants in the testtubes in the dark. In petri dishes containing MS salt, glucose 30 g/l,2,4-D 2 mg/l, and bactoagar 8 g/l, calluses were induced from the whitesprouts and grown at 25° C. in the growth chamber under the controlledconditions.

[0031] In order to achieve transduction by use of Agrobacterium, as manyas 200 seeds (Oryza sativa cv. Nakdong) were removed of their hulls andthen treated with 70% ethanol for 1 min with gentle agitation. Followingdecantation of the ethanol, the seeds were sterilized again in 10 ml of20% clorax for 1 min with gentle agitation and then washed with sterilewater. The induction of calluses, and the selection of transformedcalluses subsequent to the co-cultivation thereof together withAgrobacterium were performed in the same manner as disclosed (Hiei Y. etal., 1994, Plant J. 6:271-282) except that ?phosphyinotrysin was addedat amounts of 7 mg/l and 4 mg/l to the selection medium and theregeneration medium, respectively.

EXAMPLE 3 GFP Fluorometry

[0032] To determine the excitation and emission spectra of the GFP thatthe transgenic plants produced, water-soluble proteins were extractedfrom leaves of the transgenic rice which had been grown for threemonths.

[0033] To this end, first, sliced leaves were homogenized in anextraction buffer (20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 30 mM NaCl, 2 mMphenylmethanesulfonyl fluoride). After the centrifugation of thehomogenate at 12,000×g for 10 min, a portion of the supernatant thusobtained was added to the extraction buffer to make a final volume of 1ml. Using an assay kit (Bio-Rad) according to manufacturer'sinstruction, water-soluble proteins were quantitatively analyzed withbovine serum albumin serving as a control.

[0034] At room temperature, a quantitative measurement was made of theGFP fluorescence of cell extracts with the aid of an F-450 fluorometer(Hitachi, Japan) in 10 mm/10 mm cuvettes. After passing through theexcitation and emission monochromator used in the present invention, thelight had a bandpass of 5 nm. As for the emission spectrum, it was readat a fixed excitation ultrahigh wavelength (488) and a fixed emissionhigh wavelength (510 nm).

[0035] Results of the fluorometry are given in FIG. 2. As seen in FIG.2, the spectrum of the transgenic rice is different from that of thewild-type rice: the blue-green excitation light (480 nm) of GFP wasfound to cause a green fluorescence peak at around 510 nm. Accordingly,it was demonstrated that the distinctive green fluorescence of thetransgenic seed was originated from the product of the sgfp gene.

EXAMPLE 4 Visualization of Image from GFP Fluorescence

[0036] It is certain that, if the expression of a gene of interest isvisualized from a live body per se, great advances will be made possiblein molecular biological and biochemical research for gene expression,signal transduction, cell division, and protein location.

[0037] In order to monitor gene expression in a rice plant, a jellyfishgene encoding GFP was used as a marker. To this end, the GFP gene wasattached to an Act1 promoter. After being transformed with a recombinantplasmid harboring the GFP gene downstream of the Act1 promoter, calluseswere grown to sprout under light, and finally, seeds were obtained. Fromtransgenic plants in each developmental stage, the fluorescenceresulting from the expression of GFP was detected. Image data from thetransgenic plants was compared with that from wild type plants.

[0038] A digital video imaging system comprising a halogen lamp,bandpass filters, a CCD camera equipped with a zoom lens, and a computerwas arranged as shown in FIG. 3 to take images of plants by use of theGFP fluorescence therefrom. The light generated from a 250W halogen lampwas passed through a blue bandpass filter (Edmund, USA) to produceexcitation light having a peak at 480 nm. This filtered light wasprojected to a plant sample at an angle of 45° to the axis vertical tothe horizontal plane of the sample. The illumination area was set to be4 cm² at a maximum photon flux density (PFD) of 2 μmole m⁻²s⁻¹. Ahigh-resolution CCD color video camera (Roper Scientific Inc., USA,Model CoolSNAP) equipped with a zoom lens (Nikon, Japan) was positionedon the axis vertical to the plane of the sample to take a photograph ofthe sample while focusing on a certain area of 20 mm×20 mm of the sampleby use of the zoom lens. A bandpass filter for passing green light wasestablished in front of the CCD camera to pass only the greenfluorescent light having a peak at 510 nm. The light passing through theblue bandpass filter and the green bandpass filter had peaks at 480 nmand 510 nm, respectively, as shown in FIG. 4. The images taken by theCCD camera were transmitted through an interface board (Roper ScientificInc., USA) to a personal computer (Pentium III CPU at 500 MHz, IntelCorp., USA) and the image data was stored therein. Image results areshown in FIGS. 5 to 7.

[0039] Referring to FIG. 5, there are two fluorographs of calluses whichwere of wild type (A) and transformed with a GFP gene (B), respectively.As seen in these fluorographs, the characteristic green fluorescenceobserved in the transformed callus is found to result from theexpression of GFP in the callus because no fluorescence was observed inthe wild type callus. In detecting GFP fluorescence, the redautofluorescence of chlorophyll usually acts as an inhibitory factor.However, the system comprising suitable bandpass filters, a sensitiveCCD camera, and a relatively intense light beam could overcome thebarrier.

[0040] With reference to FIG. 6, fluorographs of rice sprouts are shown.Wild type sprouts exhibited no fluorescence (left panel A and C) whilebright green fluorescence was detected from the whole organs oftransgenic white and green sprouts (right panel B and D).

[0041]FIG. 7 shows fluorographs taken from rice seeds which are a wildtype (A) and transgenic (B and C). No fluorescence was observed from thewild type rice seed (A). On the other hand, the expression of GFP intransgenic rice seeds was detected whether they were shelled or not (Band C).

INDUSTRIAL APPLICABILITY

[0042] As described hereinbefore, the method and system of the presentinvention requires no additional gene products, substrates, norsubsidiary factors in visualizing the expression of GFP in various plantorgans and tissues, including calluses, sprouts, and seeds. In addition,the method and system of the present invention can detect in vivo GFPexpression very quickly and easily, making great contribution toresearch into gene expression in various plant tissues and organs, anddevelopmental traits, as well as the selection of transgenic seeds.

[0043] The present invention has been described in an illustrativemanner, and it is to be understood that the terminology used is intendedto be in the nature of description rather than of limitation. Manymodifications and variations of the present invention are possible inlight of the above teachings. Therefore, it is to be understood thatwithin the scope of the appended claims, the invention may be practicedotherwise than as specifically described.

1. A fluorometry method for monitoring the transformation of plantsbased on the in vivo expression of a heterologous green fluorescentprotein, comprising the steps: projecting excitation light from a lightsource through a blue bandpass filter onto a plant sample at an angle of45°, said blue bandpass filter passing light ranging, in wavelength,from 470 to 490, said excitation light having a wavelength of around 488nm with a peak at 480 nm; detecting light generated from the plantsample by use of a charge coupled device color video camera equippedwith a zoom lens, which is positioned on the axis vertical to the planeof the sample, said light passing through a green bandpass filter whichpasses light ranging, in wavelength, from 500 to 550 nm before arrivingat said zoom lens, so as to have a wavelength of around 509 nm, andphotographing the image of the plant sample on the basis of the lightgenerated from the plant sample; and processing the image in a computerto determine whether the plant sample is transgenic or not.
 2. A systemfor monitoring the transformation of plants on the basis of the in vivoexpression of a heterologous green fluorescent protein, utilizing themethod of claim 1.